1. Conformational changes in apolipoprotein B modulate intracellular assembly and degradation of ApoB-containing lipoprotein particles in HepG2 cells.
- Author
-
Macri J and Adeli K
- Subjects
- Animals, Apolipoprotein B-100, Apolipoproteins B drug effects, Apolipoproteins B metabolism, Carcinoma, Hepatocellular metabolism, Carcinoma, Hepatocellular pathology, Cystine drug effects, Dithiothreitol pharmacology, Glycosylation drug effects, Humans, Leupeptins pharmacology, Lipoproteins chemistry, Lipoproteins, HDL chemistry, Lipoproteins, HDL metabolism, Lipoproteins, LDL chemistry, Lipoproteins, LDL metabolism, Lipoproteins, VLDL chemistry, Lipoproteins, VLDL metabolism, Liver Neoplasms metabolism, Liver Neoplasms pathology, Microsomes, Liver metabolism, Molecular Weight, Neoplasm Proteins chemistry, Neoplasm Proteins drug effects, Neoplasm Proteins metabolism, Protease Inhibitors pharmacology, Protein Denaturation, Protein Folding, Protein Processing, Post-Translational drug effects, Sulfhydryl Reagents pharmacology, Tumor Cells, Cultured metabolism, Tunicamycin pharmacology, Apolipoproteins B chemistry, Lipoproteins metabolism, Liver metabolism, Protein Conformation drug effects
- Abstract
The linkage between the conformation of apolipoprotein B100 (apoB) and the intracellular assembly and degradation of apoB-containing lipoproteins was investigated in the present study. Disruption of disulfide bond formation in newly synthesized apoB molecules through the use of the reducing agent DTT resulted in a decrease in the secretion of apoB-containing lipoproteins from HepG2 cells compared with control cells. The synthesis of total apoB (apoB100 plus nascent chains), as well as a number of control proteins, such as albumin and alpha 1-antitrypsin, was decreased significantly in DTT-treated cells. However, the intracellular accumulation of full-length apoB100 molecules was not inhibited in the presence of DTT. Subcellular fractionation indicated that apoB molecules isolated from the microsomes of DTT-treated cells had an increased association with the microsomal membrane compared with apoB isolated from untreated cells. Analysis of the distribution of apoB-containing lipoproteins from the lumen of isolated microsomes demonstrated that in the presence of DTT, there was a shift in the distribution, such that there was a decrease in the formation of HDL-sized (lipid-poor) apoB-containing lipoproteins and a decrease in the formation of LDL/VLDL apoB particles. Alterations in apoB conformation and their impact on degradation were also investigated by using DTT and by inhibiting N-linked glycosylation with tunicamycin. DTT appeared to change the rate and pattern of apoB degradation. Degradation was accelerated in both intact and permeabilized HepG2 cells. ApoB degradation occurred in DTT-treated permeabilized cells without the usual generation of the 70-kD and 335-kD fragments and was largely N-acetyl-leucyl-leucyl-norleucinal (ALLN) insensitive. In tunicamycin-treated cells, DTT further accelerated the degradation of unglycosylated apoB. Overall, the data suggest that the misfolding of apoB may prevent the proper association of apoB with lipids, resulting in impairment of the assembly of mature apoB-containing lipoproteins. Alteration in the conformation of apoB also appears to alter the degradation pathway of apoB, such that the protein is degraded through a pathway that is at least in part ALLN insensitive.
- Published
- 1997
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